The ultimate goal of these studies is to determine the role of intestinal peptide hydrolases in human nutrition and what role, if any, deficiencies of these enzymes play in human diseases. Substrates highly discriminating for the 5 peptide-hydrolyzing enzymes recently identified in human intestinal brush border will be used to determine the levels of these enzyme activities in intestinal biopsies of normal subjects and of patients with various intestinal diseases of undetermined etiology. Protein-separating techniques including ion-exchange chromatography, acrylamide gel electrophoresis, and affinity chromatography will be used to separate and identify each peptide hydrolase present in the cytosol of human intestinal mucosa. Biochemical characteristics and substrates specificity of each isolated enzyme will be determined. Particular emphasis will be placed on determining substrates which are highly discriminating for each enzyme isolated. Various protein-separating techniques including immunoaffinity chromatography will be employed to purify one or more of the 5 human intestinal brush border peptide hydrolyzing enzymes. Careful substrate specificity studies of each purified enzyme will be made. The metal ion cofactor for rat intestinal brush border aminopeptidase IIB will be determined by activation studies of the apoenzyme formed by removal of metal from the native enzyme and by metal analysis of the purified enzyme. Leucine chloromethylketone, a strong inhibitor of rat brush border enzyme IIB, will be used in peptide absorption experiments to determine the relative importance of brush border hydrolysis versus intact absorption for uptake of various peptides.